Cell viability was assayed using the crystal violet method as described (12 (link),13 (link),28 (link)). Subconfluent HUVECs were seeded in 96-well plates (10,000 cells/well). After 16 hr of culture, cells were treated with 1.25, 2.5, 5, 10, or 20 μM of Kyn (Cat #: K8625) or ILA (Cat #: I5508) in ECMb supplemented with 0.2% heated inactivated FBS and 1% p/s up to 2 days (5 wells/treatment). Stock solutions of Kyn and ILA were prepared in dimethyl sulfoxide (DMSO, Cat # D2650). Additional cells were treated with the vehicle control (DMSO, 0.02% v/v; the maximum concentration in the final Kyn and ILA solutions). Kyn, ILA, and DMSO were all purchased from Sigma-Aldrich, St. Louis, MO, USA. Media were changed with ECMb containing Kyn, ILA, or DMSO daily. At the end of treatment, the optical density value of each well was measured using a microplate reader at 570 nm (Biotek, Winooski, VT, USA).
Protocol detail
Crystal Violet Assay for Cell Viability
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Corresponding Organization :
Other organizations : Meriter Hospital, University of Wisconsin–Madison, UnityPoint Health Meriter, Shanghai First Maternity and Infant Hospital, Guangdong Medical College
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Variable analysis
independent variables
- Kyn (Cat #: K8625) at concentrations of 1.25, 2.5, 5, 10, or 20 μM
- ILA (Cat #: I5508) at concentrations of 1.25, 2.5, 5, 10, or 20 μM
- Cell viability measured by optical density at 570 nm
- Cell type: Subconfluent HUVECs
- Seeding density: 10,000 cells/well in 96-well plates
- Culture duration: Up to 2 days
- Culture media: ECMb supplemented with 0.2% heated inactivated FBS and 1% p/s
- Media change: Daily
- Negative control: Vehicle control (DMSO, 0.02% v/v)
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